1. Field of the art
This invention relates to a novel nucleotide derivative. More particularly, the present invention pertains to a nucleotide derivative in which a primary amino group has been introduced through an alkylene group on extension from the 3'-phosphate group of deoxycytidine, and which is bound to a carrier through a spacer to the amino group of cytosine.
2. Prior art
I have previously proposed oligonucleotide derivatives in which an aminoalkyl group has been introduced at 5'-ends (hereinafter referred to as 5'-end aminated oligonucleotide) (Japanese Patent Laid-Open Publications Nos. 93098/1984, 93099/1984 and 93100/1984). The 5'-aminated oligonucleotide can be bound to various labeling materials (or simply labels) and carriers through the amino group introduced. Examples of the labeling material may include biotin, 2,4-dinitrobenzene, fluorescent substances (rhodamine, fluorescein, etc.), enzyme proteins (horse radish peroxidase, alkali phosphatase, .beta.-galactosidase) and metalated proteins (ferrictin, etc.). Among these, concerning biotin and 2,4-dinitrobenzene, I have already proposed oligonucleotide derivatives having these bound thereto and methods for preparation thereof (Japanese Patent Laid-Open Publication No. 148798/1984 and Japanese Patent Application No. 75878/1983). On the other hand, as the carrier, Sepharose, etc. may be considered, and the oligonucleotide derivatives bound to carriers (hereinafter called immobilized nucleotide) and preparation thereof have already been proposed by me (Japanese Patent Laid-Open Publication No. 27900/1984).
Examples of the uses of immobilized nucleotides may include isolation and purification of mRNA in general, isolation of mRNA having specific base sequence [J. Biochem., 81, 941 (1977)], separation of double-stranded DNA, isolation and purification of single-stranded DNA, synthesis of long chain oligonucleotides and isolation and purification of nucleic acid-concerned enzymes.
Also, oligonucleotide derivatives having as a label biotin or 2,4-dinitrobenzene bound thereto can be used as a non-radioactive affinity probe for detection of target genes, nucleic acid bindable proteins, etc., and utilized for the biotin-avidin technique [DNA, 3, 269-277 (1984), Nucleic Acids Research, 5, 363-384 (1978)], the enzyme immunoassay [Nucleic Acids Research, 10, 6789-6796 (1982), etc.], the fluorescent antibody technique [Nucleic Acids Research, 12, 1791-1810] and the electron microscope technique [the above Nucleic Acids Research, etc.]. See BIOMEDICAL BUSINESS INTERNATIONAL, 7. 78-79 (1984), Nature, 306, 5941 (1983), DNA, 2, 72 (1983), etc.
Thus, since oligonucleotides and derivatives thereof are of very great potential utilization value (Journal "BIOTECHNOLOGY", AUGUST (1983), published by NATURE PUBLISHING COMPANY), the establishment of a method of synthesizing the same and preparation of novel derivatives thereof are being urgently sought.
Accordingly, I have also proposed, subsequent to the above 5'-end aminated oligonucleotides, oligonucleotide derivatives in which an amino alkyl group has been introduced at the 3'-end (hereinafter called 3'-end aminated oligonucleotide) (Japanese Patent Applications Nos. 22474/1984 and 22475/1984). For the 3'-end aminated oligonucleotide, various uses may be considered similarly as for the above 5'-end aminated oligonucleotide, and it is also possible to utilize it in combination with the 5'-end aminated oligonucleotide as a compound forming a pair. However, since the 3'-end aminated oligonucleotide can be synthesized only by the liquid phase method, the following problems have been encountered.
(1) The reaction scale is large.
(2) In the respective steps of oligonucleotide synthesis (deprotection, condensation, etc.), purification of intermediates is required, and, additionally, the skill of an expert is required for the operation of oligonucleotide synthesis. As a result, much synthesis time and labor are necessary.
Thus, the development of a method for solving these various problems has been greatly desired.